HIV requires the host cell machinery for replication. Many complexes hijacked by HIV have been identified, but structures are known in only a few cases. The HARC Center is taking a broad systems-to-structure approach to this problem, having identified and validated new host complexes through a comprehensive proteomics effort. Primary biological aims of the Center are to achieve comprehensive structural pictures of: (1) how the accessory proteins Vif, Vpu, Vpr, and the viral protease PR disarm host defenses and circumvent viral restriction via degradation pathways, and (2) how the regulatory proteins Tat and Rev hijack the host transcription and RNA trafficking machinery to express and package viral RNAs. There is growing evidence that HIV accessory proteins primarily target host antiviral restriction factors for destruction. For Vif, we will determine the structures of the E3 ligase complex and interactions with APOBEC, and evaluate the functional roles of the CBF? cofactor and post-translational modifications (PTMs). For Vpu, we will determine the structures of restriction factor complexes and map effects of Vpu on ubiquitination. For Vpr, we will validate new host interactions and mechanisms and assemble complexes for structure determination. For PR, we will determine the structures and functions of new host target complexes and measure the levels of PR activity during infection. The regulatory proteins Tat and Rev hijack host transcription and RNA export machineries. For Tat, we will determine the structures of newly discovered AFF4 elongation complexes, and characterize other host factors, PTMs, and inhibitory 7SK snRNP complexes. For Rev, we will determines the structures of Rev-RRE nuclear export complexes, map viral RNA structures, and characterize the roles of new host proteins in post export functions. The HARC Center also relies on technology innovation from four cores. The EM Core will develop methods to determine structures of membrane protein complexes, and use Fabs to solve the structures of small HIV-host complexes. The Proteomics Core will extend mass spectrometry analyses to host protein complexes and map HIV-dependent host PTMs. The Computational Core will develop methods to characterize allostery and detailed models to study PTMs. The Virology Core will measure effects of new host interactions on HIV replication and coupled activities of Tat and Rev. The individual projects and technologies depend critically on an extensive network of collaborators, which will be expanded through a Collaborative Opportunity Fund. PUBLIC HEALTH RELEVANCE: Existing anti-HIV therapeutics have extended the life expectancy of infected individuals, however major limitations remain, including drug resistance. By determining structures of key HIV accessory and regulatory complexes, we will achieve a more complete molecular understanding of how the virus hijacks the host cell machinery, and also identify new targets for therapeutic intervention in the continuing battle against AIDS.